Fluorescence lifetime measurements and biological imaging. Mikhail Y. Berezin and Samuel Achilefu. Washington University School of Medicine, St. Louis, USA. This review discusses fluorescence lifetime, an intrinsic property of fluorophores, which is not affected by measurement methods or environmental factors. It is a key parameter in fluorescence lifetime imaging (FLIM), which can be used to monitor functional changes and enhance imaging contrast. FLIM is versatile, applicable to various phases and systems, and has broad applications in materials science, arts, aeronautics, agriculture, forensics, biology, and medicine. The history of fluorescence lifetime dates back to the mid-19th century, with early studies on phosphorescence and the development of instruments like the phosphoroscope. The Kerr cell was later used to measure ultrafast time intervals, leading to the development of FLIM. The review covers the theory of fluorescence lifetime, measurement techniques, and factors affecting it, including temperature, viscosity, and polarity. It also discusses internal and external quenching processes, such as internal rotation and electron/proton transfer, which influence fluorescence lifetime. The review highlights the importance of fluorescence lifetime in biological imaging and its applications in various fields.Fluorescence lifetime measurements and biological imaging. Mikhail Y. Berezin and Samuel Achilefu. Washington University School of Medicine, St. Louis, USA. This review discusses fluorescence lifetime, an intrinsic property of fluorophores, which is not affected by measurement methods or environmental factors. It is a key parameter in fluorescence lifetime imaging (FLIM), which can be used to monitor functional changes and enhance imaging contrast. FLIM is versatile, applicable to various phases and systems, and has broad applications in materials science, arts, aeronautics, agriculture, forensics, biology, and medicine. The history of fluorescence lifetime dates back to the mid-19th century, with early studies on phosphorescence and the development of instruments like the phosphoroscope. The Kerr cell was later used to measure ultrafast time intervals, leading to the development of FLIM. The review covers the theory of fluorescence lifetime, measurement techniques, and factors affecting it, including temperature, viscosity, and polarity. It also discusses internal and external quenching processes, such as internal rotation and electron/proton transfer, which influence fluorescence lifetime. The review highlights the importance of fluorescence lifetime in biological imaging and its applications in various fields.